Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α-synuclein in PC12 cells

Wang,Runqing; Zhao,Jie; Zhang,Jiewen; Liu,Wei; Zhao,Meiying; Li,Jiangtao; Lv,Juan; Li,Yanan

doi:10.3892/etm.2015.2432

Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α-synuclein in PC12 cells

Authors:
- Runqing Wang
- Jie Zhao
- Jiewen Zhang
- Wei Liu
- Meiying Zhao
- Jiangtao Li
- Juan Lv
- Yanan Li
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Affiliations: Department of Neurology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, Henan 450007, P.R. China, Department of Neurology, Henan Provincial People's Hospital, Zhengzhou, Henan 450003, P.R. China
Published online on: April 20, 2015 https://doi.org/10.3892/etm.2015.2432
Pages: 2088-2094
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of this study was to investigate the effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α‑synuclein, and to analyze its role in the pathogenesis of Parkinson's disease (PD). PC12 cells subjected to nerve growth factor‑induced differentiation were used as the cell model to study the dopaminergic neurons, and the lysosomal and proteasomal inhibitors trans‑epoxysuccinyl‑L‑leucylamido‑(4‑guanidino) butane (E64) and, respectively, were used exclusively and in combination to treat the PC12 cells. The viability and metabolic state of the cells was assessed using the MTT assay; flow cytometry was used to measure the rate of cell apoptosis; and the double immunofluorescence method was applied to observe the formation of thioflavin S- and α‑synuclein protein‑positive aggregates and inclusion bodies in the PC12 cells. In addition, the Hoechst 33258 staining method was used to observe the apoptosis of the α‑synuclein protein and thioflavin‑S double‑labeled cells. Following the administration of the lysosomal and proteasomal pathway inhibitors, the cell viability decreased in a concentration‑dependent manner and the cell apoptosis rate increased. The proportion of PC12 cells with α‑synuclein protein‑positive aggregates and inclusion bodies in the E64 group was 7.94%, compared with 20.33 and 36.77% in the lactacystin and combination treatment groups, respectively. Statistical analysis indicated that the number of inclusion body‑positive cells in the treatment groups was significantly higher than that in the control group (3.78%) (P<0.05). Apoptosis was evident in the double‑positive cells with α‑synuclein protein‑positive inclusion bodies (17.29±1.54%). In conclusion, lysosomal and proteasomal dysfunction may play an important role in the pathogenesis of PD through the induction of abnormal α-synuclein protein aggregation in dopaminergic neurons.

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